Graft materials containing ECM components, and methods for their manufacture

ABSTRACT

Described are packaged, sterile medical graft products containing controlled levels of a growth factor such as Fibroblast Growth Factor-2 (FGF-2). Also described are methods of manufacturing medical graft products wherein processing, including sterilization, is controlled and monitored to provide medical graft products having modulated, known levels of a extracellular matrix factor, such as a growth factor, e.g. FGF-2. Preferred graft materials are extracellular matrix materials isolated from human or animal donors, particularly submucosa-containing extracellular matrix materials. Further described are ECM compositions that are or are useful for preparing gels, and related methods for preparation and use.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/650,647 filed Jan. 8, 2007, which is acontinuation of U.S. patent application Ser. No. 11/435,393 filed May16, 2006, which is a continuation-in-part of U.S. patent applicationSer. No. 10/569,218, which is a National Stage of InternationalApplication No. PCT/US2004/027557 filed Aug. 25, 2004, which claims thebenefit of U.S. Patent Application Ser. No. 60/497,746 filed Aug. 25,2003, each of which is hereby incorporated by reference in its entirety.This application also claims the benefit of priority of U.S. ProvisionalPatent Application Ser. Nos. 60/681,522, 60/681,278, 60/681,689 and60/681,511, all filed May 16, 2005, each of which is hereby incorporatedby reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to materials useful for tissuegrafting, and in particular to such materials derived from extracellularmatrices and retaining both collagen and substances such as growthfactors that contribute to the beneficial properties of the materials.In one aspect, the invention relates to extracellular matrix tissuegraft materials containing one or more growth factors modulated to apredetermined level, and related methods of manufacturing.

Extracellular matrix (ECM) materials, including those derived fromsubmucosa and other tissues, are known tissue graft materials. See,e.g., U.S. Pat. Nos. 4,902,508, 4,956,178, 5,281,422, 5,372,821,5,554,389, 6,099,567, and 6,206,931. Tissues from various biologicalstructures can be used for these purposes, including for example smallintestine, stomach, the urinary bladder, skin, pericardium, dura mater,fascia, and the like. These sources provide collagenous materials usefulin a variety of surgical procedures where tissue support and/or ingrowthare desired.

Submucosa and other ECM materials have been shown to include a varietyof components other than collagen that that can contribute to thebioactivity of the materials and to their value in medical grafting andother uses. As examples, ECM materials can include growth factors, celladhesion proteins, and proteoglycans. However, ECM materials aretypically subjected to a battery of manipulations in the manufacture offinished products containing them. This presents challenges in obtainingfinished products that not only possess the necessary physicalproperties and appropriate levels of biocompatibility and sterility, butalso the desired bioactivity. The present invention is addressed tothese needs.

SUMMARY

Accordingly, in one aspect, the present invention provides a method formanufacturing a tissue graft material such as a collagenousextracellular matrix containing at least one extractable, bioactivegrowth factor or other non-collagenous protein material, particularlyFibroblast Growth Factor-2 (FGF-2), at a predetermined amount. Themethod includes the steps of providing a non-sterile extracellularmatrix material; fashioning a plurality of graft products from theextracellular matrix material; packaging the products; subjecting thepackaged products to a sterilization procedure that affects the level ofextractable bioactive growth factor (FGF-2) or other non-collagenousprotein material in the products; and, taking and testing sampleproducts of the sterilized packaged products to determine a level of agrowth factor (FGF-2) in the sample products, wherein said determinedlevel is representative of an approximate level of said growth factor inother ones of said products from the lot from which the sample productwas taken.

In another aspect, the present invention provides a medical product thatcomprises a packaged, sterile animal-derived extracellular matrixmaterial comprising FGF-2 at a level of at least about 50 nanograms pergram dry weight. Particularly preferred materials are lyophilized and/orinclude submucosa.

Another aspect of the invention provides a packaged, sterileextracellular matrix material isolated from animal tissue and includingcomponents native to the tissue, the matrix material including collagen,growth factors, proteoglycans, glycosaminoglycans, and havingextractable, bioactive FGF-2 at a level of at least about 50 nanogramsper gram dry weight.

Another aspect of the invention provides a method for manufacturing asterile, extracellular matrix material. The method includes isolating anextracellular matrix material from animal tissue, the isolatedextracellular matrix material including extractable FGF-2 at a firstlevel; and, sterilizing the isolated extracellular matrix material underconditions to retain the extractable, bioactive FGF-2 in at least 10% ofthe first level.

Another aspect of the invention provides a method for manufacturingmedical products. The method includes providing extracellular matrixmaterial in non-sterile condition and isolated from animal tissue, theextracellular matrix material comprising extractable, bioactive FGF-2;packaging and sterilizing the extracellular matrix material to provideproduct lots each containing multiple, packaged extracellular matrixmaterial products; taking sample products from the product lots; andtesting the sample products to determine whether they includeextractable, bioactive FGF-2 at a level above a predetermined level,e.g. above about 50 nanograms per gram dry weight.

Another aspect of the invention provides a medical product adapted fortreating wounds, the product including an extracellular matrix materialisolated from animal tissue, the material including bioactive componentsuseful to treat wounds including but not limited to FGF-2. The FGF-2 ispresent in the extracellular matrix material at a level of at leastabout 50 nanograms per gram dry weight.

Another aspect of the invention relates to a medical product comprisinga dry collagenous powder comprising extracellular matrix material,wherein the dry collagenous powder is effective to gel upon rehydrationwith an aqueous medium and comprises FGF-2 at a level of at least about50 ng/g dry weight.

In another aspect, the invention relates to a medical product comprisinga fluid composition comprising solubilized or suspended collagenousextracellular matrix material, wherein the fluid composition comprisesFGF-2 at a level of about 0.1 ng/ml to about 100 ng/ml.

In another embodiment, the invention provides a method for disinfectingan aqueous extracellular matrix hydrolysate composition. The aqueousextracellular matrix hydrolysate composition is contacted with anoxidizing disinfectant for a period of time and under conditionssufficient to disinfect the aqueous extracellular matrix hydrolysatecomposition.

Another aspect of the invention relates to a method for preparing adisinfected, extracellular matrix hydrolysate composition. This methodcomprises forming an aqueous extracellular matrix hydrolysate. A firstdialysis step is conducted and includes dialyzing the aqueousextracellular matrix hydrolysate against an aqueous medium containing anoxidizing disinfectant so as to contact and disinfect the extracellularmatrix hydrolysate with the oxidizing disinfectant and thereby form adisinfected extracellular matrix hydrolysate. A second dialysis stepincludes dialyzing the disinfected extracellular matrix hydrolysateunder conditions to remove the oxidizing disinfectant.

In another embodiment, the invention provides an extracellular matrixhydrolysate product having extracellular matrix components disinfectedby contact of an aqueous medium containing the extracellular matrixhydrolysate with an oxidizing disinfectant. The extracellular matrixhydrolysate product can take on a variety of forms, including a drypowdery material, a non-gelled aqueous composition, a gel, or a sponge.

Still another embodiment of the invention provides an extracellularmatrix graft material that includes an extracellular matrix hydrolysatecombined with extracellular matrix particles. In a preferred form, thegraft material includes an aqueous medium having said extracellularmatrix hydrolysate in a dissolved state with the extracellular matrixparticles suspended therein, desirably wherein the medium exhibitsgel-forming capacity.

Another embodiment of the invention provides an extracellular matrixgraft material that includes a sterile, injectable fluid extracellularmatrix composition including an aqueous medium containing anextracellular matrix hydrolysate. The extracellular matrix hydrolysateis present in the composition at a level of at least about 20 mg/ml, forexample in the range of about 20 mg/ml to about 200 mg/ml.

Additional aspects as well as features and advantages of the inventionwill be apparent to those of ordinary skill in the art from thedescriptions herein.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to certain embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications in the illustrated device, and such further applicationsof the principles of the invention as described herein beingcontemplated as would normally occur to one skilled in the art to whichthe invention relates.

As disclosed above, in one aspect, the present invention providespackaged, sterile medical products including tissue grafts materialscontaining one or more growth factors, and methods for manufacturing thesame. As for the tissue graft material used, it will desirably be anaturally-derived material such as an extracellular matrix (ECM)material. Preferred are naturally-derived collagenous ECMs isolated fromsuitable animal or human tissue sources. Suitable extracellular matrixmaterials include, for instance, submucosa (including for example smallintestinal submucosa, stomach submucosa, urinary bladder submucosa, oruterine submucosa, each of these isolated from juvenile or adultanimals), renal capsule membrane, amnion, dura mater, pericardium,serosa, peritoneum or basement membrane materials, including liverbasement membrane or epithelial basement membrane materials. Thesematerials may be isolated and used as intact natural sheet forms, orreconstituted collagen layers including collagen derived from thesematerials and/or other collagenous materials may be used. For additionalinformation as to submucosa materials useful in the present invention,and their isolation and treatment, reference can be made to U.S. Pat.Nos. 4,902,508, 5,554,389, 5,993,844, 6,206,931, and 6,099,567. Renalcapsule membrane can also be obtained from warm-blooded vertebrates, asdescribed more particularly in International Patent Application serialNo. PCT/US02/20499 filed Jun. 28, 2002, published Jan. 9, 2003, asWO03002165.

Preferred ECM base materials contain residual bioactive proteins orother ECM components derived from the tissue source of the materials.For example, they may contain Fibroblast Growth Factor-2 (basic FGF),Transforming Growth Factor-beta (TGF-beta) and vascular endothelialgrowth factor (VEGF). It is also expected that ECM base materials of theinvention may contain additional bioactive components including, forexample, one or more of glycosaminoglycans, glycoproteins,proteoglycans, and/or growth factors.

It has been discovered that the sterilization conditions utilized in themanufacture of tissue graft materials can significantly impact the levelof one or more of such bioactive components or growth factors, includingfor example FGF-2. Accordingly, in accordance with the invention,sterilization protocols can be selected and controlled to modulate thelevel of growth factors, for example by either intentionally reducinggrowth factor levels to a predetermined level or below, or to retain atleast a given percentage or level of one or more growth factors,particularly FGF-2, in the material. In certain embodiments of theinvention, the ECM or other graft material is processed to finished,packaged, sterile products containing FGF-2 at a level of at least 50ng/g dry weight, or even at least about 60, at least about 70, at leastabout 80, or at least about 100 ng/g dry weight. In other embodiments ofthe invention, an ECM material will have a first level of a bioactivecomponent, such as FGF-2 or another growth factor, after isolation fromthe animal or human donor source tissue and rinsing with a rinse agentsuch as water. The ECM material will thereafter be processed undercontrolled conditions, including sterilization, to provide packaged,sterile medical products containing at least about 10% of said firstlevel of the FGF-2 or other bioactive component, or even at least 15%,20%, 30% or even 50% or more of said first level.

Illustratively, it has been found that sterilization protocols includingethylene oxide (EO) sterilization, electron beam (E-beam) radiation andgas plasma sterilization (e.g. Sterrad®) can significantly reduce levelsof extractable, bioactive FGF-2. At the same time, these sterilizationtechniques have significantly lower or essentially no impact on levelsof extractable, bioactive TGF-beta. Advantageously, the modulation ofgrowth factors imparted by the sterilization technique can be used toaffect and optimize levels of given growth factors, their ratios, etc.,to prepare a graft material better suited for a particular medicalindication wherein the retained growth factor or growth factors arebeneficial to the indication, and/or wherein eliminated growth factor orgrowth factors are deleterious to the medical indication.

For example, FGF-2 is known to stimulate angiogenesis, neurite growth,plasminogen activator (PA) secretion, and matrix metalloproteinase 1(MMP-1) production. Correspondingly, levels of FGF-2 can be retained andoptimized for use in the graft material in wound healing (angiogenesis),treatment of nervous tissue (neurite outgrowth) including peripheralnervous tissue and central nervous tissue, modulating adhesion formation(by stimulating PA), and facilitating collagen turnover and degradation(by stimulating MMP-1 production). Thus, FGF-2 levels can be retained inthe material as high as possible by selecting and optimizing thesterilization protocol. For instance, it has been found that non-sterileisolated submucosa layers (and in particular isolated from smallintestine), contain relatively high levels of extractable, bioactiveFGF-2. For example, submucosa tissue isolated from small intestine andminimally treated, e.g. only by rinsing, may be recovered so as tocontain in excess of about 100 nanograms per gram of FGF-2 dry weightand potentially even higher levels such as above about 200 or about 400nanograms per gram. In manufacturing, it may be beneficial to retain asmuch of this FGF-2 in the material as possible. Thus, intermediate stepsbetween the isolation of the original submucosa material and thefinished, packaged medical article, can be selected and controlled so asto maintain as much active FGF-2 in the material as possible.

As one example, an isolated, small intestinal submucosa materialdisinfected as described in U.S. Pat. No. 6,206,931 with peracetic acidmay contain from about 70 to about 200 nanograms per gram (dry weight)of FGF-2. It has been found that sterilization treatments using ethyleneoxide, E-beam, and gas plasma sterilization techniques significantlyreduce the levels of FGF-2 in the disinfected material. Among these,E-beam sterilization had the smallest impact on FGF-2 levels, withE-beam sterilized submucosa having FGF-2 levels ranging from about 75nanograms per gram dry weight to about 150 nanograms per gram dryweight, and generally retaining greater than about 50% of the FGF-2level of the disinfected submucosa material. Gas plasma sterilizedmaterial had an FGF-2 level ranging from about 60 nanograms per gram dryweight to about 110 nanograms per gram dry weight, and retaining atleast 40% of the FGF-2 level of the disinfected submucosa material.Thus, in embodiments of the invention, materials sterilized using E-beamor gas plasma techniques are used in products configured for and methodsfor treating patients where relatively high FGF-2 levels are beneficial,for example wound healing, treatment of tissue of the nervous system,modulating adhesions, or facilitating collagen turnover and degradation.

On the other hand, ethylene oxide sterilization at both low temperatureand high temperature conditions had a more significant impact inreducing the FGF-2 levels, with products typically having from about 10to about 40 nanograms per gram of FGF-2 dry weight, and retaining lessthan about 40% of the FGF-2 level of the disinfected submucosa material(e.g. about 10% to about 40%). In this ethylene oxide work, the hightemperature conditions tended to do have a slightly greater effect inreducing the FGF-2 levels than the low temperature conditions.Accordingly, in the ethylene oxide and potentially other sterilizationtechniques, the temperature may be increased or decreased to provide arespective higher or lower level of reduction of FGF-2 and/or othergrowth factors or non-collagenous ECM proteins. Similarly, the totaldose of sterilant chemical or energy can be increased or decreased toprovide a respective higher or lower level of reduction of FGF-2 and/orother growth factors or non-collagenous ECM proteins. Increased doses ofsterilant can be achieved, for instance, through a longer, singleexposure of the graft material to the sterilant, or through multiple,discreet exposures of the graft material to the sterilant.

In accordance with the invention, in addition to controlling thesterilization protocol, a number of other manufacturing techniques canbe undertaken to provide a packaged, sterilized graft product with acontrolled level of one or more growth factors, including for exampleFGF-2. As a first measure, where it is desired to retain as high aspossible a level of FGF-2, the animal-derived collagenous ECM can beprocessed and preserved from the time of harvest to the time at whichFGF-2 or other growth factor is protected against further significantdegradation. For these purposes, the harvested tissue from which the ECMmaterial is to be isolated may be placed soon or immediately afterharvest in a stabilizing solution that prevents degradation of theproduct including for example, osmotic, hypoxic, autolytic, and/orproteolytic degradation. This solution can also protect againstbacterial contamination. To achieve these effects, the stabilizingmaterial may be a buffered solution of anti-oxidants, antibiotics,protease inhibitors, oncotic agents, or other stabilizing agents.

Illustratively, enzymes (e.g. superoxide dismutase and catalase) may beused to neutralize the superoxide anion and hydrogen peroxide orcompounds that can directly react with and neutralize other free-radicalspecies. Antioxidants may be added and include tertiarybutylhydroquinone (BHT), alpha tocopherol, mannitol, hydroxyurea,glutathione, ascorbate, ethylenediaminetetraacetic acid (EDTA) and theamino acids histidine, proline and cysteine. In addition toantioxidants, the stabilizing solution may contain agents to inhibithypoxic alteration to normal biochemical pathways, for example,allopurinol to inhibit xanthine dehydrogenase, lipoxigenase inhibitors,calcium channel blocking drugs, calcium binding agents, iron bindingagents, metabolic intermediaries and substrates of adenosinetriphosphate (ATP) generation.

The stabilizing solution may also contain one or more antibiotics,antifungal agents, protease inhibitors, proteoglycans, and anappropriate buffer. Antibiotics can be used to inhibit or preventbacterial growth and subsequent tissue infection. Antibiotics may beselected from the group of penicillin, streptomycin, gentamicin,kanamycin, neomycin, bacitracin, and vancomycin. Additionally,anti-fungal agents may be employed, including amphotericin-B, nystatinand polymyxin.

Protease inhibitors may be included in the stabilizing solution toinhibit endogenous proteolytic enzymes which, when released, can causeirreversible degradation of the ECM, as well as the release ofchemoattractant factors. These chemoattractants solicit the involvementof polymorphonuclear leukocytes, macrophages and other natural killercells which generate a nonspecific immune response that can furtherdamage the ECM. Protease inhibitors can be selected from the groupconsisting of N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride(PMSF), ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),leupeptin, ammonium chloride, elevated pH and apoprotinin.

Glycosaminoglycans may be included in the stabilizing solution toprovide a colloid osmotic balance between the solution and the tissue,thereby preventing the diffusion of endogenous glycosaminoglycans fromthe tissue to the solution. Endogenous glycosaminoglycans serve avariety of functions in collagen-based connective tissue physiology.They may be involved in the regulation of cell growth anddifferentiation (e.g. heparin sulfate and smooth muscle cells) or,alternatively, they are important in preventing pathologicalcalcification (as with heart valves). Glycosaminoglycans are alsoinvolved in the complex regulation of collagen and elastin synthesis andremodeling, which is fundamental to connective tissue function.Glycosaminoglycans are selected from the group of chondroitin sulfate,heparin sulfate, and dermatan sulfate and hyaluronan.Non-glycosaminoglycan osmotic agents which may also be included arepolymers such as dextran and polyvinyl pyrolodone (PVP) and amino acidssuch as glycine and proline.

The stabilizing solution can also contain an appropriate buffer. Thenature of the buffer is important in several aspects of the processingtechnique. Crystalloid, low osmotic strength buffers have beenassociated with damage occurring during saphenous vein procurement andwith corneal storage. Optimum pH and buffering capacity against theproducts of hypoxia damage (described below), is essential. In thiscontext the organic and bicarbonate buffers have distinct advantages.(In red cell storage, acetate-citrate buffers with glycine and glucosehave been shown to be effective in prolonging shelf-life and maintainingcellular integrity.) The inventors prefer to use an organic bufferselected from the group consisting of 2-(N-morpholino)ethanesulfonicacid (MES), 3-(N-morpholine)propanesulfonic acid (MOPS) andN-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid (HEPES).Alternatively, a low salt or physiological buffer, including phosphate,bicarbonate and acetate-citrate, may be more appropriate in certainapplications.

In another aspect, components of the stabilizing solution address one ormore of the events that occur during the harvesting of tissues, such asspasm, hypoxia, hypoxia reperfusion, lysosomal enzyme release, plateletadhesion, sterility and buffering conditions. Involuntary contraction ofthe smooth muscles can result from mechanical stretching or distension,as well as from the chemical action of certain endothelial cell derivedcontraction factors, typically released under hypoxic (low oxygen)conditions. This involuntary contraction may result in damage to theadjacent ECM. For this reason, the stabilizing solution can include oneor more smooth muscle relaxants, selected from the group of calcitoningene related peptide (CGRP), papaverine, sodium nitroprusside (NaNP), H7(a protein Kinase C inhibitor) calcium channel blockers, calciumchelators, isoproterenol, phentolamine, pinacidil,isobutylmethylxanthine (IBMX), nifedepine and flurazine. The harvestedtissue can be immediately placed into this stabilizing solution and ismaintained at 4° C. during transportation and any storage prior tofurther processing.

The tissue graft material of the invention can be provided in anysuitable form, including substantially two-dimensional sheet form(optionally meshed sheet), or a three-dimensional form such as a tube,valve leaflet, or the like. The tissue graft material may contain asingle layer of isolated ECM material, or may be a multilaminateconstruct sized the same as its component layers (e.g. containingdirectly overlapped layers) or larger than its component layers (e.g.containing partially overlapped layers), see, e.g., U.S. Pat. Nos.5,885,619 and 5,711,969.

In another embodiment, the invention provides a medical product thatincludes a dry collagenous powder useful for example to treat wounds orto otherwise induce tissue growth at a desired implant location, andincluding an ECM material. The powder is desirably effective to gel uponrehydration with an aqueous medium and includes FGF-2 at a level of atleast about 50 nanograms per gram dry weight. Illustratively, the powdercan include a particulate of ECM material prepared by drying a fluidizedmaterial prepared as described in U.S. Pat. Nos. 5,516,533 and5,275,826. This resulting powder can be used alone or in combinationwith other powder materials to support gelling of the overall powderupon rehydration with an aqueous medium such as a buffered salinesolution. In this regard, in addition to the particulate ECM material,the powder composition may also include powdered, purified collagen,gelatin, or the like, to assist in gelling the product upon rehydration.

In one embodiment, the preparation of the powder will be conducted toinclude FGF-2 at a level of at least about 50 nanograms per gram dryweight, more preferably at least about 60, 70, 80, or 100 nanograms pergram dry weight. Resultant fluid compositions containing solubilized orsuspended collagenous ECM materials will desirably be prepared tocontain FGF-2 at a level of about 0.1 nanograms per milliliter orgreater, e.g. typically in the range of about 0.1 nanograms per ml toabout 100 nanograms per ml. This fluid composition is desirably gelable,for example upon incubation for a time after rehydration, which may behastened by bringing the fluid composition to a relatively neutral pHand/or to body temperature from room temperature. In other embodiments,the fluidized medical product may contain FGF-2 at a level of about 1 toabout 15 nanograms per ml, or may contain FGF-2 at a level of about 10to about 30 nanograms per ml.

As disclosed above, certain embodiments of the invention providepackaged, sterile medical products. Known packaging techniques andmaterials can be used in the manufacture of such products, with thepackaging being selected to suit the final sterilization technique beingemployed, e.g. ethylene oxide gas, electron-beam, or gas plasmatechniques. In addition, the packaging may contain or otherwise bearindicia relating to the use of the enclosed graft material for aparticular medical indication, e.g. wound care, and/or may contain orotherwise bear indicia as to one or more growth factors (e.g. FGF-2) forwhich the product manufacture has been controlled to modulate its level,e.g. to reflect a minimum level of such growth factor, a maximum levelof such growth factor, or a range of such growth factor contained in theenclosed tissue graft product.

In other embodiments, the present invention provides ECM gelcompositions and methods and materials for their preparation, which canoptionally also be used in conjunction with the techniques describedabove for modulating the level of one or more bioactive substances inthe product, including for example growth factors such as FGF-2. The gelcompositions of the invention can be prepared from an isolated ECMmaterial, for example one of those listed above. The ECM material isused to prepare a solubilized mixture including components of thematerial. This can be achieved by digestion of the ECM material in anacidic or basic medium and/or by contact with an appropriate enzyme orcombination of enzymes.

Typically, the ECM material is reduced to particulate form to aid in thedigestion step. This can be achieved by tearing, cutting, grinding orshearing the isolated ECM material. Illustratively, shearing may beconducted in a fluid medium, and grinding may be conducted with thematerial in a frozen state. For example, the material can be contactedwith liquid nitrogen to freeze it for purposes of facilitating grindinginto powder form. Such techniques can involve freezing and pulverizingsubmucosa under liquid nitrogen in an industrial blender.

Any suitable enzyme may be used for an enzymatic digestion step. Suchenzymes include for example serine proteases, aspartyl proteases, andmatrix metalloproteases. The concentration of the enzyme can be adjustedbased on the specific enzyme used, the amount of submucosa to bedigested, the duration of the digestion, the temperature of thereaction, and the desired properties of the final product. In oneembodiment about 0.1% to about 0.2% of enzyme (pepsin, for example) isused and the digestion is conducted under cooled conditions for a periodof time sufficient to substantially digest the ECM material. Thedigestion can be conducted at any suitable temperature, withtemperatures ranging from 4-37° C. being preferred. Likewise, anysuitable duration of digestion can be used, such durations typicallyfalling in the range of about 2-180 hours. The ratio of theconcentration of ECM material (hydrated) to total enzyme can be anysuitable ration, for instance ranging from about 25 to about 2500; incertain aspects, this ration usually ranges from about 25 to about 125and more typically the ratio is about 50, and the digestion is conductedat 4° C. for 24-72 hours. When an enzyme is used to aid in thedigestion, the digestion will be performed at a pH at which the enzymeis active and more advantageously at a pH at which the enzyme isoptimally active. Illustratively, pepsin exhibits optimal activity atpH's in the range of about 2-4.

The enzymes or other disruptive agents used to solubilize the ECMmaterial can be removed or inactivated before or during the gellingprocess so as not to compromise gel formation or subsequent gelstability. Also, any disruptive agent, particularly enzymes, that remainpresent and active during storage of the tissue will potentially changethe composition and potentially the gelling characteristics of thesolution. Enzymes, such as pepsin, can be inactivated with proteaseinhibitors, a shift to neutral pH, a drop in temperature below 0° C.,heat inactivation or through the removal of the enzyme by fractionation.A combination of these methods can be utilized to stop digestion of theECM material at a predetermined endpoint, for example the ECM materialcan be immediately frozen and later fractionated to limit digestion.

The ECM material is enzymatically digested for a sufficient time toproduce a hydrolysate of ECM components. The ECM can be treated with oneenzyme or with a mixture of enzymes to hydrolyze the structuralcomponents of the material and prepare a hydrolysate having multiplehydrolyzed components of reduced molecular weight. The length ofdigestion time is varied depending on the application, and the digestioncan be extended to completely solubilize the ECM material. In some modesof operation, the ECM material will be treated sufficiently to partiallysolubilize the material to produce a digest composition comprisinghydrolyzed ECM components and nonhydrolyzed ECM components. The digestcomposition can then optionally be further processed to remove at leastsome of the nonhydrolyzed components. For example, the nonhydrolyzedcomponents can be separated from the hydrolyzed portions bycentrifugation, filtration, or other separation techniques known in theart.

Preferred gel compositions of the present invention are prepared fromenzymatically digested vertebrate ECM material that has beenfractionated under acidic conditions, for example including pH rangingfrom about 2 to less than 7, especially to remove low molecular weightcomponents. Typically, the ECM hydrolysate is fractionated by dialysisagainst a solution or other aqueous medium having an acidic pH, e.g. apH ranging from about 2 to about 5, more desirably greater than 3 andless than 7. In addition to fractionating the hydrolysate under acidicconditions, the ECM hydrolysate is typically fractionated underconditions of low ionic strength with minimal concentrations of saltssuch as those usually found in standard buffers such as PBS (i.e., NaCl,KCl, Na₂HPO₄, or KH₂PO₄) that can pass through the dialysis membrane andinto the hydrolysate. Such fractionation conditions work to reduce theionic strength of the ECM hydrolysate and thereby provide enhanced gelforming characteristics.

The hydrolysate solution produced by enzymatic digestion of the ECMmaterial has a characteristic ratio of protein to carbohydrate. Theratio of protein to carbohydrate in the hydrolysate is determined by theenzyme utilized in the digestion step and by the duration of thedigestion. The ratio may be similar to or may be substantially differentfrom the protein to carbohydrate ratio of the undigested ECM tissue. Forexample, digestion of vertebrate ECM material with a protease such aspepsin, followed by dialysis, will form a fractionated ECM hydrolysatehaving a lower protein to carbohydrate ratio relative to the originalECM material.

In accordance with certain embodiments of the invention, shape retaininggel forms of ECM are prepared from ECM material that has beenenzymatically digested and fractionated under acidic conditions to forman ECM hydrolysate that has a protein to carbohydrate ratio differentthan that of the original ECM material. Such fractionation can beachieved entirely or at least in part by dialysis. The molecular weightcut off of the ECM components to be included in the gel material isselected based on the desired properties of the gel. Typically themolecular weight cutoff of the dialysis membrane (the molecular weightabove which the membrane will prevent passage of molecules) is within inthe range of about 2000 to about 10000 Dalton, and more preferably fromabout 3500 to about 5000 Dalton. In once such embodiment, thedigested/solubilized extracellular matrix composition is dialyzedagainst an acidic solution having a low ionic strength. For example, thesolubilized extracellular matrix composition can be dialyzed against ahydrochloric acid solution, but any other acids including acetic acid,formic acid, lactic acid, citric acid, sulfuric acid, ethanoic acid,carbonic acid, nitric acid, or phosphoric acid can be used. In variousillustrative embodiments, the fractionation, for example by dialysis,can be performed at about 2° C. to about 37° C. for about 1 hour toabout 96 hours. In another illustrative embodiment, and theconcentration of the acid, such as acetic acid, hydrochloric acid,formic acid, lactic acid, citric acid, sulfuric acid, ethanoic acid,carbonic acid nitric acid, or phosphoric acid, against which thesolubilized extracellular matrix composition is dialyzed can be fromabout 0.001 N to about 0.1 N, from about 0.005 N to about 0.1 N, fromabout 0.01 N to about 0.1 N, from about 0.05 N to about 0.1 N, fromabout 0.001 N to about 0.05 N, about 0.001 N to about 0.01 N, or fromabout 0.01 N to about 0.05 N. In one illustrative embodiment, thesolubilized extracellular matrix composition can be dialyzed against0.01 N HCl. However, the fractionation can be performed at anytemperature, for any length of time, and against any concentration ofacid. In one embodiment of the invention, apart from the potentialremoval of undigested ECM components after the digestion step and anycontrolled fractionation to remove low molecular weight components asdiscussed above, the ECM hydrolysate is processed so as to avoid anysubstantial further physical separation of the ECM components. Forexample, when a more concentrated ECM hydrolysate material is desired,this can be accomplished by removing water from the system (e.g. byevaporation or lyophilization) as opposed to using conventional “saltingout”/centrifugation techniques that would demonstrate significantselectivity in precipitating and isolating collagen, leaving behindamounts of other desired ECM components. Thus, in certain embodiments ofthe invention, solubilized ECM components of the ECM hydrolysate remainsubstantially unfractionated, or remain substantially unfractionatedabove a predetermined molecular weight cutoff such as that used in thedialysis membrane, e.g. above a given value in the range of about 2000to 10000 Dalton, more preferably about 3500 to about 5000 Dalton.

In another embodiment, the source extracellular matrix material can beextracted in addition to being solubilized with an acid and/or treatedwith an enzyme. Extraction methods for extracellular matrices are knownto those skilled in the art and are described in detail in U.S. Pat. No.6,375,989, incorporated herein by reference. Illustrative extractionexcipients include, for example, chaotropic agents such as urea,guanidine, sodium chloride or other neutral salt solutions, magnesiumchloride, and non-ionic or ionic surfactants.

Vertebrate ECM material can be stored frozen (e.g. at about −20 to about−80° C.) in either its solid, comminuted or enzymatically digested formsprior to formation of the gel compositions of the present invention, orthe material can be stored after being hydrolyzed and fractionated. TheECM material can be stored in solvents that maintain the collagen in itsnative form and solubility. For example, one suitable storage solvent is0.01 M acetic acid, however other acids can be substituted, such as 0.01N HCl. In accordance with one embodiment the fractionated ECMhydrolysate is dried (by lyophilization, for example) and stored in adehydrated/lyophilized state. The dried form can be rehydrated andgelled to form a gel of the present invention.

In this regard, an ECM hydrolysate as described herein (fractionated orunfractionated) can be dried to form a powder or other dried solidcomposition in any suitable manner. In certain embodiments a liquidmedium containing the ECM hydrolysate is dried under lyophilizationconditions or other similar conditions that involve the removal of waterand/or another hydrant by sublimation from solid to gas. In one aspect,a solubilized extracellular matrix composition is lyophilized after anenzymatic digestion as described herein. In another aspect, asolubilized extracellular matrix composition is lyophilized after anenzymatic digestion and subsequent separation step that removes anyinsoluble components. In yet another aspect, the solubilizedextracellular matrix composition is lyophilized after digestion andfractionation steps as described herein. In another embodiment, apolymerized matrix formed with a solubilized extracellular matrixcomposition as described herein is lyophilized. In one illustrative modeof carrying out lyophilization steps as described herein, thesolubilized extracellular matrix composition is first frozen, and thenplaced under a vacuum. In another lyophilization embodiment, thesolubilized extracellular matrix composition is freeze-dried under avacuum. Any method of lyophilization known to the skilled artisan can beused.

If the solubilized extracellular matrix composition is lyophilized, thelyophilized extracellular matrix composition can be stored frozen or atroom temperature (for example, at about −80° C. to about 25° C.).Storage temperatures are selected to stabilize the solubilizedextracellular matrix components. The compositions can be stored forabout 1-26 weeks, or longer. In one illustrative embodiment, alyophilized or otherwise dried extracellular matrix composition willinclude an acid as a storage excipient, for example deriving from anacid present in an aqueous or other liquid medium containing thesolubilized extracellular matrix composition that is subjected tolyophilization or other drying conditions. In certain embodiments,hydrochloric acid is included in the lyophilized or otherwise driedextracellular matrix composition. As examples, hydrochloric acid, orother acids, at concentrations of from about 0.001 N to about 0.1 N,from about 0.005 N to about 0.1 N, from about 0.01 N to about 0.1 N,from about 0.05 N to about 0.1 N, from about 0.001 N to about 0.05 N,from about 0.001 N to about 0.01 N, or from about 0.01 N to about 0.05 Ncan be used as a storage excipient for the lyophilized/dried,solubilized extracellular matrix components. Other acids can be used asstorage excipients including for instance acetic acid, formic acid,lactic acid, citric acid, sulfuric acid, ethanoic acid, carbonic acid,nitric acid, or phosphoric acid, and these acids can be used at any ofthe above-described concentrations. In one illustrative embodiment, alyophilizate or other dried material can be prepared from an acidicmedium, such as an acetic acid medium, at a concentration of from about0.001 M to about 0.5 M or from about 0.01 M to about 0.5 M. In anotherembodiment, a lyophilizate or other dried material can be prepared froman aqueous medium with a pH of about 6 or below. In other illustrativeembodiments, lyoprotectants, cryoprotectants, lyophilizationaccelerators, or crystallizing excipients (e.g., ethanol, isopropanol,mannitol, trehalose, maltose, sucrose, tert-butanol, and tween 20), orcombinations thereof, and the like can be present during lyophilization.

In accordance with one embodiment, the fractionated ECM hydrolysate willexhibit the capacity to gel upon adjusting the pH of a relatively moreacidic aqueous medium containing it to about 5 to about 9, morepreferably about 6.6 to about 8.0, and typically about 7.2 to about 7.8,thus inducing fibrillogenesis and matrix gel assembly. In oneembodiment, the pH of the fractionated hydrolysate is adjusted by theaddition of a buffer that does not leave a toxic residue, and has aphysiological ion concentration and the capacity to hold physiologicalpH. Examples of suitable buffers include PBS, HEPES, and DMEM. In oneembodiment the pH of the fractionated ECM hydrolysate is raised by theaddition of a buffered NaOH solution to 6.6 to 8.0, more preferably 7.2to 7.8. Any suitable concentration of NaOH solution can be used forthese purposes, for example including about 0.05 M to about 0.5 M NaOH.In accordance with one embodiment, the ECM hydrolysate is mixed with abuffer and sufficient 0.25 N NaOH is added to the mixture to achieve thedesired pH. If desired at this point, the resultant mixture can bealiquoted into appropriate forms or into designated cultureware andincubated at 37° C. for 0.5 to 1.5 hours to form an ECM gel.

The ionic strength of the ECM hydrolysate is believed to be important inmaintaining the fibers of collagen in a state that allows forfibrillogenesis and matrix gel assembly upon neutralization of thehydrolysate. Accordingly, if needed, the salt concentration of the ECMhydrolysate material can be reduced prior to neutralization of thehydrolysate. The neutralized hydrolysate can be caused to gel at anysuitable temperature, e.g. ranging from about 4° C. to about 40° C. Thetemperature will typically affect the gelling times, which may rangefrom 5 to 120 minutes at the higher gellation temperatures and 1 to 8hours at the lower gellation temperatures. Typically, the hydrolysatewill be gelled at elevated temperatures to hasten the gelling process,for example at 37° C. In this regard, preferred neutralized ECMhydrolysates will be effective to gel in less than about ninety minutesat 37° C., for example approximately thirty to ninety minutes at 37° C.Alternatively, the gel can be stored at 4° C., and under theseconditions the setting of the gel will be delayed, e.g. for about 3-8hours.

Additional components can be added to the hydrolysate compositionbefore, during or after forming the gel. For example, proteinscarbohydrates, growth factors, therapeutics, bioactive agents, nucleicacids, cells or pharmaceuticals can be added. In certain embodiments,such materials are added prior to formation of the gel. This may beaccomplished for example by forming a dry mixture of a powdered ECMhydrolysate with the additional component(s), and then reconstitutingand gelling the mixture, or by incorporating the additional component(s)into an aqueous, ungelled composition of the ECM hydrolysate before,during (e.g. with) or after addition of the neutralization agent. Inother embodiments, the additional component(s) are added to the formedECM gel, e.g. by infusing or mixing the component(s) into the gel and/orcoating them onto the gel.

In one embodiment of the invention, a particulate ECM material will beadded to the hydrolysate composition, which will then be incorporated inthe formed gel. Such particulate ECM materials can be prepared bycutting, tearing, grinding or otherwise comminuting an ECM startingmaterial. For example, a particulate ECM material having an averageparticle size of about 50 microns to about 500 microns may be includedin the hydrolysate, more preferably about 100 microns to about 400microns. The ECM particulate can be added in any suitable amountrelative to the hydrolysate, with preferred ECM particulate to ECMhydrolysate weight ratios (based on dry solids) being about 0.1:1 toabout 200:1, more preferably in the range of 1:1 to about 100:1. Theinclusion of such ECM particulates in the ultimate gel can serve toprovide additional material that can function to provide bioactivity tothe gel (e.g. itself including FGF-2 and/or other growth factors orbioactive substances as discussed herein) and/or serve as scaffoldingmaterial for tissue ingrowth.

In certain embodiments, a solubilized extracellular matrix compositionas described herein can be sterilized in any suitable manner. Exemplarysterilizing agents and techniques are described above, but anysterilizing agent or method of sterilization known in the art can beused. For example, a solubilized extracellular matrix composition can besterilized using glutaraldehyde, formaldehyde, acidic pH, propyleneoxide, ethylene oxide, gas plasma sterilization, gamma radiation,electron beam sterilization, or peracetic acid sterilization, orcombinations thereof, and the like. Sterilization techniques which donot adversely affect the structure and biotropic properties of thecomponents of the solubilized extracellular matrix composition can beused.

In certain embodiments, an ECM hydrolysate material to be used in tissueaugmentation, e.g. in functional or cosmetic purposes, will incorporatean ECM particulate material. In these embodiments, the ECM particulatematerial can be included at a size and in an amount that effectivelyretains an injectable character to the hydrolysate composition, forexample by injection through a needle having a size in the range of 18to 31 gauge (internal diameters of 0.047 inches to about 0.004 inches).In this fashion, non-invasive procedures for tissue augmentation will beprovided, which in preferred cases will involve the injection of anungelled ECM hydrolysate containing suspended ECM particles at arelatively lower (e.g. room) temperature, which will be promoted to forma gelled composition when injected into a patient and thereby brought tophysiologic temperature (about 37° C.).

In other aspects of the invention, it has been discovered thatprocessing techniques that involve contacting the ECM material with adisinfecting oxidizing agent compound can significantly affect not onlythe concentration of bioactive substances but also the gelling qualityof the collagen molecules. In particular, it has been found thatcontacting an ECM material with an oxidizing agent such as peraceticacid prior to digestion to form the ECM hydrolysate can disrupt orimpair the ability of ECM hydrolysate to form a gel. On the other hand,contacting an aqueous medium including ECM hydrolysate components withan oxidizing disinfectant such as a peroxy compound provides an improvedability to recover a disinfected ECM hydrolysate that exhibits thecapacity to form beneficial gels. In accordance with one embodiment ofthe invention, an aqueous medium containing ECM hydrolysate componentsis disinfected by providing a peroxy disinfectant in the aqueous medium.This is advantageously achieved using dialysis to deliver the peroxydisinfectant into and/or to remove the peroxy disinfectant from theaqueous medium containing the hydrolysate. In one preferred embodiment,the aqueous medium containing the ECM hydrolysate is dialyzed against anaqueous medium containing the peroxy disinfectant to deliver thedisinfectant into contact with the ECM hydrolysate, and then is dialyzedagainst an appropriate aqueous medium (e.g. an acidic aqueous medium) toat least substantially remove the peroxy disinfectant from the ECMhydrolysate. During this dialysis step, the peroxy compound passesthrough the dialysis membrane and into the ECM hydrolysate, and contactsECM components for a sufficient period of time to disinfect the ECMcomponents of the hydrolysate. In this regard, typical contact timeswill range from about 0.5 hours to about 8 hours and more typicallyabout 1 hour to about 4 hours. The period of contact will be sufficientto substantially disinfect the digest, including the removal ofendotoxins and inactivation of virus material present. The removal ofthe peroxy disinfectant by dialysis may likewise be conducted over anysuitable period of time, for example having a duration of about 4 toabout 180 hours, more typically about 24 to about 96 hours. In oneembodiment, the sterilized, solubilized extracellular matrix compositioncan be dialyzed against 0.01 N HCl, for example, prior to lyophilizationto remove the sterilization solution and so that the solubilizedextracellular matrix components are in a 0.01 N HCl solution whenlyophilized. Alternatively, the solubilized extracellular matrixcomposition can be dialyzed against acetic acid, for example, prior tolyophilization and can be lyophilized in acetic acid and redissolved inHCl or water. In general, the disinfection step will desirably result ina disinfected ECM hydrolysate composition having sufficiently low levelsof endotoxins, viral burdens, and other contaminant materials to renderit suitable for medical use. Endotoxin levels below about 2 endotoxinunits (EUs) per gram (dry weight) are preferred, more preferably belowabout 1 EU per gram, as are virus levels below 100 plaque forming unitsper gram (dry weight), more preferably below 1 plaque forming unit pergram.

In one embodiment, the aqueous ECM hydrolysate composition is asubstantially homogeneous solution during the dialysis step fordelivering the oxidizing disinfectant to the hydrolysate compositionand/or during the dialysis step for removing the oxidizing disinfectantfrom the hydrolysate composition. Alternatively, the aqueous hydrolysatecomposition can include suspended ECM hydrolysate particles, optionallyin combination with some dissolved ECM hydrolysate components, duringeither or both of the oxidizing disinfectant delivery and removal steps.Dialysis processes in which at least some of the ECM hydrolysatecomponents are dissolved during the disinfectant delivery and/or removalsteps are preferred and those in which substantially all of the ECMhydrolysate components are dissolved are more preferred.

The disinfection step can be conducted at any suitable temperature, andwill typically be conducted between 0° C. and 37° C., more typicallybetween about 4° C. and about 15° C. During this step, the concentrationof the ECM hydrolysate solids in the aqueous medium is typically in therange of about 2 mg/ml to about 200 mg/ml, and may vary somewhat throughthe course of the dialysis due to the migration of water through themembrane. In certain embodiments of the invention, a relativelyunconcentrated digest is used, having a starting ECM solids level ofabout 5 mg/ml to about 15 mg/ml. In other embodiments of the invention,a relatively concentrated ECM hydrolysate is used at the start of thedisinfection step, for example having a concentration of at least about20 mg/ml and up to about 200 mg/ml, more preferably at least about 100mg/ml and up to about 200 mg/ml. It has been found that the use ofconcentrated ECM hydrolysates during this disinfection processingresults in an ultimate gel composition having higher gel strength thanthat obtained using similar processing with a lower concentration ECMhydrolysate. Accordingly, processes which involve the removal of amountsof water from the ECM hydrolysate resulting from the digestion prior tothe disinfection processing step are preferred. For example, suchprocesses may include removing only a portion of the water (e.g. about10% to about 98% by weight of the water present) prior to thedialysis/disinfection step, or may include rendering the digest to asolid by drying the material by lyophilization or otherwise,reconstituting the dried material in an aqueous medium, and thentreating that aqueous medium with the dialysis/disinfection step.

Certain impacts of dialysis processing conditions upon ECM hydrolysategels are illustrated in specific work to date described moreparticularly in Examples 2-5 below. Generally, several differentsubmucosa hydrolysates were prepared while varying the acid presentduring pepsin digestion and varying the concentration of ECM hydrolysatepresent during dialysis against a peracetic acid (PAA) solution.Specifically, a first gel (A1) was prepared using 0.5 M acetic acid inthe pepsin digestion solution, and about 5-15 mg/ml ECM hydrolysateduring the PAA disinfection; a second gel (A2) was prepared using 0.5 Macetic acid in the pepsin digestion solution, and about 130-150 mg/mlECM hydrolysate during the PAA disinfection; a third gel (H1) wasprepared using 0.01 M hydrochloric acid in the pepsin digestionsolution, and about 5-15 mg/ml ECM hydrolysate during the PAAdisinfection; and a fourth gel (H2) was prepared using 0.01 Mhydrochloric acid in the pepsin digestion solution, and about 130-150mg/ml ECM hydrolysate during the PAA disinfection. The processed ECMhydrolysates were provided in a solution of 0.1 M HCl at a concentrationof about 30 mg/ml, and then PBS was added and the pH of the mixture wasadjusted to 7.5-7.6 with 0.25 M NaOH to gel the composition. Themechanical properties of the various gels were then assessed. Theresults are summarized in Table 1 below.

TABLE 1 Gel Compressive Modulus (kPa) Compressive Strength (kPa A1 1 0.5A2 7 2 H1 10 3 H2 20 7

As can be seen, the gels prepared using high submucosa hydrolysateconcentrations during the disinfection step (A2,H2) were relativelystronger than those prepared using low submucosa hydrolysateconcentrations (A1, H1). In addition, in cell growth assays, the A2 andH2 gels demonstrated an improved capacity to support the proliferationof primary human dermal fibroblast and primary human bladder smoothmuscle cells as compared to the A1 and H1 gels. In other observations,the gels prepared from ECM hydrolysate materials resultant of HCl/pepsindigestion were relatively stronger than the corresponding gels resultantof acetic acid/pepsin digestion. Thus, the conditions used during thepreparation and processing of ECM hydrolysate materials can be selectedand controlled to modulate the physical and biological properties of theultimate ECM gel compositions.

In one mode of operation, the disinfection of the aqueous mediumcontaining the ECM hydrolysate can include adding the peroxy compound orother oxidizing disinfectant directly to the ECM hydrolysate, forexample being included in an aqueous medium used to reconstitute a driedECM hydrolysate or being added directly to an aqueous ECM hydrolysatecomposition. The disinfectant can then be allowed to contact the ECMhydrolysate for a sufficient period of time under suitable conditions(e.g. as described above) to disinfect the hydrolysate, and then removedfrom contact with the hydrolysate. In one embodiment, the oxidizingdisinfectant can then be removed using a dialysis procedure as discussedabove. In other embodiments, the disinfectant can be partially orcompletely removed using other techniques such as chromatographic or ionexchange techniques, or can be partially or completely decomposed tophysiologically acceptable components. For example, when using anoxidizing disinfectant containing hydrogen peroxide (e.g. hydrogenperoxide alone or a peracid such as peracetic acid), hydrogen peroxidecan be allowed or caused to decompose to water and oxygen, for examplein some embodiments including the use of agents that promote thedecomposition such as thermal energy or ionizing radiation, e.g.ultraviolet radiation.

In another mode of operation, the oxidizing disinfectant can bedelivered into the aqueous medium containing the ECM hydrolysate bydialysis and processed sufficiently to disinfect the hydrolysate (e.g.as described above), and then removed using other techniques such aschromatographic or ion exchange techniques in whole or in part, orallowed or caused to decompose in whole or in part as discussedimmediately above.

Peroxygen compounds that may be used in the disinfection step include,for example, hydrogen peroxide, organic peroxy compounds, and preferablyperacids. Such disinfecting agents are used in a liquid medium,preferably a solution, having a pH of about 1.5 to about 10.0, moredesirably about 2.0 to about 6.0. As to peracid compounds that can beused, these include peracetic acid, perpropioic acid, or perbenzoicacid. Peracetic acid is the most preferred disinfecting agent forpurposes of the present invention.

When used, peracetic acid is desirably diluted into about a 2% to about50% by volume of alcohol solution, preferably ethanol. The concentrationof the peracetic acid may range, for instance, from about 0.05% byvolume to about 1.0% by volume. Most preferably, the concentration ofthe peracetic acid is from about 0.1% to about 0.3% by volume. Whenhydrogen peroxide is used, the concentration can range from about 0.05%to about 30% by volume. More desirably the hydrogen peroxideconcentration is from about 1% to about 10% by volume, and mostpreferably from about 2% to about 5% by volume. The solution may or maynot be buffered to a pH from about 5 to about 9, with more preferredpH's being from about 6 to about 7.5. These concentrations of hydrogenperoxide can be diluted in water or in an aqueous solution of about 2%to about 50% by volume of alcohol, most preferably ethanol. Additionalinformation concerning preferred peroxy disinfecting agents can be foundin discussions in U.S. Pat. No. 6,206,931, which is herein incorporatedby reference.

In one illustrative embodiment, a digested/solubilized extracellularmatrix composition is directly sterilized, for instance before anysolids separation step, for example, with peracetic acid or withperacetic acid and ethanol (e.g., by the addition of 0.18% peraceticacid and 4.8% ethanol to the solubilized extracellular matrixcomposition before the separation step). In another embodiment,sterilization can be carried out during a fractionation step. Forexample, a solubilized extracellular matrix composition can be dialyzedagainst chloroform, peracetic acid, or a solution of peracetic acid andethanol to disinfect or sterilize the solubilized extracellular matrixcomposition. Illustratively, the solubilized extracellular matrixcomposition can be sterilized by dialysis against a solution ofperacetic acid and ethanol (e.g., 0.18% peracetic acid and 4.8%ethanol). The chloroform, peracetic acid, or peracetic acid/ethanol canbe removed prior to lyophilization, for example by dialysis against anacid, such as 0.01 N HCl. In an alternative embodiment, the lyophilizedcomposition can be sterilized directly after rehydration, for example,by the addition of 0.18% peracetic acid and 4.8% ethanol. In thisembodiment, the sterilizing agent can be removed prior to polymerizationof the solubilized extracellular matrix components to form fibrils.

ECM gel materials of the present invention can be prepared to havedesirable properties for handling and use. For example, fluidized ECMhydrolysates can be prepared in an aqueous medium, which can thereafterbe caused or allowed to form of a gel. Such prepared aqueous mediums canhave any suitable level of ECM hydrolysate therein for subsequent gelformation. Typically, the ECM hydrolysate will be present in the aqueousmedium to be gelled at a concentration of about 2 mg/ml to about 200mg/ml, more typically about 20 mg/ml to about 200 mg/ml, and in somepreferred embodiments about 30 mg/ml to about 120 mg/ml. In preferredforms, the aqueous ECM hydrolysate composition to be gelled will have aninjectable character, for example by injection through a needle having asize in the range of 18 to 31 gauge (internal diameters of about 0.047inches to about 0.004 inches).

If the solubilized extracellular matrix composition is lyophilized orotherwise dried, the resulting dried material can be redissolved in anysuitable liquid medium. In certain embodiments, the dried material canbe redissolved in an acidic solution or in water. The lyophilizate orother dried material can be redissolved in, for example, acetic acid,hydrochloric acid, formic acid, lactic acid, citric acid, sulfuric acid,ethanoic acid, carbonic acid, nitric acid, or phosphoric acid, at any ofthe above-described concentrations, or can be redissolved in water. Inone illustrative embodiment, the lyophilizate or other dried material isredissolved in 0.01 N HCl. For use in producing engineered ECM-basedmatrices that can be injected in vivo or used for other purposes invitro, the redissolved lyophilizate can be subjected to varyingconditions (e.g., pH, phosphate concentration, temperature, buffercomposition, ionic strength, and composition and concentration ofsolubilized extracellular matrix composition components (dry weight/ml))that result in polymerization to form engineered ECM-based matrices forspecific tissue graft applications.

Furthermore, gel compositions can be prepared so that in addition toneutralization, heating to physiologic temperatures (such as 37° C.)will substantially reduce the gelling time of the material. As well,once the material is gelled, it can optionally be dried to form a spongesolid material. It is contemplated that commercial products mayconstitute any of the these forms of the ECM gel composition, e.g. (i)packaged, sterile powders which can be reconstituted in an acidic mediumand neutralized and potentially heated to form a gel, (ii) packaged,sterile aqueous compositions including solubilized ECM hydrolysatecomponents under non-gelling (e.g. acidic) conditions; (iii) packaged,sterile gel compositions, and (iv) packaged, sterile, dried spongecompositions; or other suitable forms. In one embodiment of theinvention, a medical kit is provided that includes a packaged, sterileaqueous composition including solubilized ECM hydrolysate componentsunder non-gelling (e.g. acidic) conditions, and a separately packaged,sterile aqueous neutralizing composition (e.g. containing a bufferand/or base) that is adapted to neutralize the ECM hydrolysate mediumfor the formation of a gel. In another embodiment of the invention, amedical kit includes a packaged, sterile, dried (e.g. lyophilized) ECMhydrolysate powder, a separately packaged, sterile aqueous acidicreconstituting medium, and a separately packaged sterile, aqueousneutralizing medium. In use, the ECM hydrolysate powder can bereconstituted with the reconstituting medium to form a non-gelledmixture, which can then be neutralized with the neutralizing medium forthe formation of the gel.

Medical kits as described above may also include a device, such as asyringe, for delivering the neutralized ECM hydrolysate medium to apatient. In this regard, the sterile, aqueous ECM hydrolysate medium orthe sterile ECM hydrolysate powder of such kits can be provided packagedin a syringe or other delivery instrument. In addition, the sterilereconstituting and/or neutralizing medium can be packaged in a syringe,and means provided for delivering the contents of the syringe into toanother syringe containing the aqueous ECM hydrolysate medium or the ECMhydrolysate powder for mixing purposes. In still other forms of theinvention, a self-gelling aqueous ECM hydrolysate composition can bepackaged in a container (e.g. a syringe) and stable against gelformation during storage. For example, gel formation of such productscan be dependent upon physical conditions such as temperature or contactwith local milieu present at an implantation site in a patient.Illustratively, an aqueous ECM hydrolysate composition that does not gelor gels only very slowly at temperatures below physiologic temperature(about 37° C.) can be packaged in a syringe or other container andpotentially cooled (including for example frozen) prior to use forinjection or other implantation into a patient.

In particular applications, ECM hydrolysate compositions that formhydrogels at or near physiologic pH and temperature will be preferredfor in vivo bulking applications, for example in the treatment of stressurinary incontinence, gastroesophageal reflux disease, cosmetic surgery,vesico urethral reflux, anal incontinence and vocal cord repair. Theseforms of the submucosa or other ECM gel have, in addition to collagen,complex extracellular matrix sugars and varying amounts of growthfactors in other bioactive agents that can serve to remodel tissue atthe site of implantation. These ECM hydrolysate compositions can, forexample, be injected into a patient for these applications.

ECM gels and dry sponge form materials of the invention prepared bydrying ECM gels can be used, for example, in wound healing and/or tissuereconstructive applications, or in the culture of cells.

Generally, it has been found that the manipulations used to prepare ECMhydrolysate compositions and gellable or gelled forms thereof can alsohave a significant impact upon growth factors or other ECM componentsthat may contribute to bioactivity. Techniques for modulating andsampling for levels of FGF-2 or other growth factors or bioactivesubstances can also be used in conjunction with the manufacture of thedescribed ECM hydrolysate compositions of the invention. Illustratively,it has been discovered that the dialysis/disinfection processes of theinvention employing peroxy compounds typically cause a reduction in thelevel of FGF-2 in the ECM hydrolysate material. In work to date asdescribed in Examples 2-5, such processing using peracetic acid asdisinfectant has caused a reduction in the level of FGF-2 in the rangeof about 30% to about 50%. Accordingly, to retain higher levels ofFGF-2, one can process for a minimal about of time necessary to achievethe desired disinfection of the material; on the other hand, to reducethe FGF-2 to lower levels, the disinfection processing can be continuedfor a longer period of time. In one embodiment of the invention, thedisinfection process and subsequent steps will be sufficiently conductedto result in a medically sterile aqueous ECM hydrolysate composition,which can be packaged using sterile filling operations. In otherembodiments, any terminal sterilization applied to the ECM hydrolysatematerial (e.g. in dried powder, non-gelled aqueous medium, gelled orsponge form) can also be selected and controlled to optimize the levelof FGF-2 or other bioactive substances in the product. Terminalsterilization methods may include, for example, high or low temperatureethylene oxide, radiation such as E-beam, gas plasma (e.g. Sterrad), orhydrogen peroxide vapor processing.

Preferred, packaged, sterilized ECM hydrolysate products prepared inaccordance with the invention will have an FGF-2 level (this FGF-2 beingprovided by the ECM hydrolysate) of about 100 ng/g to about 5000 ng/gbased upon the dry weight of the ECM hydrolysate. More preferably, thisvalue will be about 300 ng/g to about 4000 ng/g. As will be understood,such FGF-2 levels can be determined using standard ELISA tests (e.g.using the Quantikine Human Basic Fibroblast Growth Factor ELISA kitcommercially available from R&D Systems).

In order to promote a further understanding of the present invention andits features and advantages, the following specific examples areprovided. It will be understood that these examples are illustrative andare not limiting of the invention.

Example 1

Small intestinal submucosa material was harvested and disinfected withperacetic acid as described in U.S. Pat. No. 6,206,931. The submucosamaterial was lyophilized, packaged in medical packaging comprised ofpolyester/Tyvek and sterilized by various methods including ethyleneoxide (EO), gas plasma (hydrogen peroxide vapor), and E-beam radiation(20 kGy (plus/minus 2 kGy). The resultant submucosa material was frozenin liquid nitrogen and ground to a powder. The material was thenextracted with an extraction buffer containing 2M urea, 2.5 mg/mlheparin, and 50 mM Tris buffer, at pH 7.5 at 4° C. under constantstirring for 24 hours. After 24 hours, the extraction medium wastransferred to centrifuge tubes and the insoluble fraction pelleted at12000×G. The supernatant was transferred to dialysis tubing (MW cutoff3500) and dialyzed exhaustively against high purity (18 megaohm) water.Following dialysis the dialysate was centrifuged at 12000×G to removeany additional particulate matter and the resulting soluble extract waslyophilized. Prior to measurement the extract was reconstituted at 10mg/dry weight per ml in the manufacturer-provided diluent (R&D Systems).Samples were centrifuged to remove any insoluble matter. The resultingsupernatents were recovered and assayed for FGF-2 content using theQuantikine Human Basic Fibroblast Growth Factor Immunoassay (R&DSystems). The results are summarized in Table 2 below.

TABLE 2 ELISA Summary - CBI Extracted Tissues Sterilization Growthfactor range (ng/g) % of Non-sterile* NONE 100-210 100 EO (low temp)28-50 24 EO (high temp) 18-40 18 E-beam  50-150 66 Gas Plasma  30-125 49*based upon the average of 8 experiments.

As can be seen, E-beam and gas plasma sterilization had a significantlylower impact in reducing the level of extractable, bioactive FGF-2 inthe materials. On the other hand, ethylene oxide sterilization at bothlow and high temperatures had a significant impact in lowering the levelof extractable, bioactive FGF-2.

Example 2

Raw (isolated/washed but non-disinfected) porcine small intestinesubmucosa was frozen, cut into pieces, and cryoground to powder withliquid nitrogen. 50 g of the submucosa powder was mixed with one literof a digestion solution containing 1 g of pepsin and 0.5 M acetic acid.The digestion process was allowed to continue for 48-72 hours underconstant stirring at 4° C. At the end of the process, the digest wascentrifuged to remove undigested material. The acetic acid was thenremoved by dialysis against 0.01 M HCl for approximately 96 hours at 4°C. The resulting digest was transferred (without concentration) into asemipermeable membrane with a molecular weight cut off of 3500, anddialyzed for two hours against a 0.2 percent by volume peracetic acid ina 5 percent by volume aqueous ethanol solution at 4° C. This step servedboth to disinfect the submucosa digest and to fractionate the digest toremove components with molecular weights below 3500. The PAA-treateddigest was then dialysed against 0.01 M HCl for 48 hours at 4° C. toremove the peracetic acid. The sterilized digest was concentrated bylyophilization, forming a material that was reconstituted at about 30mg/ml solids in 0.01 M HCl and neutralized with phosphate buffered NaOHto a pH of about 7.5-7.6 and heated to physiologic temperature to form asubmucosa gel.

Example 3

A second acetic acid processed submucosa gel was made using a processsimilar to that described in Example 2 above, except concentrating thedigest prior to the PAA treatment. Specifically, immediately followingthe removal of acetic acid by dialysis, the digest was lyophilized todryness. A concentrated paste of the digest was made by dissolving apre-weighed amount of the lyophilized product in a known amount of 0.01M HCl to prepare a mixture having an ECM solids concentration of about50 mg/ml. The concentrated paste was then dialysed against the PAAsolution for 2 hours and then against 0.01 M HCl for removal of PAA inthe same manner described in Example 2. The digest was adjusted to about30 mg/ml solids and neutralized with phosphate buffered NaOH to a pH ofabout 7.5-7.6 and heated to physiologic temperature to form a submucosagel.

Example 4

An HCl processed submucosa gel was made using a procedure similar tothat described in Example 2, except using 0.01 M of HCl in thepepsin/digestion solution rather than the 0.5 M of acetic acid, andomitting the step involving removal of acetic acid since none waspresent. The digest was used to form a gel as described in Example 2.

Example 5

Another HCl processed submucosa gel was made using a procedure similarto that described in Example 3, except using 0.01 M of HCl in thepepsin/digestion solution rather than the 0.5 M of acetic acid, andomitting the step involving removal of acetic acid since none waspresent. The digest was used to form a gel as described in Example 3.

Example 6 Preparation of Lyophilized, Bioactive ECM Composition

Small intestinal submucosa is harvested and prepared from freshlyeuthanized pigs (Delphi Ind.) as previously disclosed in U.S. Pat. No.4,956,178. Intestinal submucosa is powderized under liquid nitrogen andstored at −80oC prior to use. Digestion and solubilization of thematerial is performed by adding 5 grams of powdered tissue to each 100ml of solution containing 0.1% pepsin in 0.01 N hydrochloric acid andincubating for 72 hours at 4° C. Following the incubation period, theresulting solubilized composition is centrifuged at 12,000 rpm for 20minutes at 4° C. and the insoluble pellet is discarded. The supernatantis dialyzed against at least ten changes of 0.01 N hydrochloric acid at4° C. (MWCO 3500) over a period of at least four days. The solubilizedfractionated composition is then sterilized by dialyzing against 0.18%peracetic acid/4.8% ethyl alcohol for about two hours. Dialysis of thecomposition is continued for at least two more days, with threeadditional changes of sterile 0.01 N hydrochloric acid per day, toeliminate the peracetic acid. The contents of the dialysis bags are thenlyophilized to dryness and stored.

Example 7 Preparation of Lyophilized, Bioactive ECM Composition

Small intestinal submucosa was harvested and prepared from freshlyeuthanized pigs (Delphi Ind.) as previously disclosed in U.S. Pat. No.4,956,178. Intestinal submucosa was powderized under liquid nitrogen andstored at −80° C. prior to use. Digestion and solubilization of thematerial was performed by adding 5 grams of powdered tissue to each 100ml of solution containing 0.1% pepsin in 0.01 N hydrochloric acid andincubating for 72 hours at 4° C. Following the incubation period, thesolubilized composition was centrifuged at 12,000 rpm for 20 minutes at4° C. and the insoluble pellet was discarded. The supernatant waslyophilized to dryness and stored.

Example 8 Preparation of Reconstituted, Bioactive ECM Composition

Immediately prior to use, lyophilized material from Example 2,consisting of a mixture of extracellular matrix components, wasreconstituted in 0.01 N HCl. To polymerize the soluble extracellularmatrix components into a 3-dimensional matrix, reconstitutedextracellular matrix solutions were diluted and brought to a particularpH, ionic strength, and phosphate concentration by the addition of aphosphate buffer and concentrated HCl and NaOH solutions. Polymerizationof neutralized solutions was then induced by raising the temperaturefrom 4° C. to 37° C. Various initiation buffers were used to make finalsolutions with the properties shown in Table 3. Ionic strength wasvaried based on sodium chloride concentration. The pH of thepolymerization reaction was controlled by varying the ratios of mono-and diabasic phosphate salts.

TABLE 3 pH I [Pi] [C] Series 1 SIS formulations 6.5 0.16 0.01 1 mg/ml7.0 0.16 0.01 1 mg/ml 7.4 0.17 0.01 1 mg/ml 8.0 0.17 0.01 1 mg/ml 8.50.17 0.01 1 mg/ml 9.0 0.17 0.01 1 mg/ml Series 2 SIS formulations 7.40.06 0.02 1 mg/ml 7.4 0.30 0.02 1 mg/ml 7.4 0.60 0.02 1 mg/ml 7.4 0.900.02 1 mg/ml 7.4 1.20 0.02 1 mg/ml 7.4 1.20 0.02 1 mg/ml Series 3 SISformulations 7.4 0.3 0.00 1 mg/ml 7.4 0.3 0.02 1 mg/ml 7.4 0.3 0.04 1mg/ml 7.4 0.3 0.06 1 mg/ml 7.4 0.3 0.08 1 mg/ml 7.4 0.3 0.11 1 mg/ml

Table 3: Engineered ECMs representing a complex mixture of interstitialECM components (SIS) were prepared at varied pH (series 1), ionicstrength (series 2), and phosphate concentration (series 3). [C]represents collagen concentration in mg/ml, [Pi] represents phosphateconcentration in M, and I represents ionic strength in M.

Example 9 Preparation of Reconstituted Bioactive Extracellular Matrices

Small intestinal submucosa was harvested and prepared from freshlyeuthanized pigs (Delphi Ind.) as previously disclosed in U.S. Pat. No.4,956,178. Intestinal submucosa was powderized under liquid nitrogen andstored at −80° C. prior to use. Digestion and solubilization of thematerial was performed by adding 5 grams of powdered tissue to each 100ml of solution containing 0.1% pepsin in 0.01 N hydrochloric acid andincubating with stirring for 72 hours at 4° C. Following the incubationperiod, the solubilized composition was centrifuged at 12,000 rpm for 20minutes at 4° C. and the insoluble pellet was discarded. The supernatantwas dialyzed extensively against 0.01 N HCl at 4° C. in dialysis tubingwith a 3500 MWCO (Spectrum Medical Industries). Polymerization of thesolubilized extracellular matrix composition was achieved by dialysisagainst PBS, pH 7.4, at 4° C. for about 48 hours. The polymerizedconstruct was then dialyzed against several changes of water at roomtemperature and was then lyophilized to dryness.

The polymerized construct had significant mechanical integrity and, uponrehydration, had tissue-like consistency and properties. In one assay,glycerol was added prior to polymerization by dialysis and matrices withincreased mechanical integrity and increased fibril length resulted.

Example 10 Preparation of Extracellular Matrix Threads

Small intestinal submucosa was harvested and prepared from freshlyeuthanized pigs (Delphi Ind.) as previously disclosed in U.S. Pat. No.4,956,178. Intestinal submucosa was powderized under liquid nitrogen andstored at −80° C. prior to use. Digestion and solubilization of thematerial was performed by adding 5 grams of powdered tissue to each 100ml of solution containing 0.1% pepsin in 0.01 N hydrochloric acid andincubating for 72 hours at 4° C. Following the incubation period, thesolubilized composition was centrifuged at 12,000 rpm for 20 minutes at4° C. and the insoluble pellet was discarded.

The solubilized extracellular matrix composition (at 4° C.) was placedin a syringe with a needle and was slowly injected into a PBS solutionat 40° C. The solubilized extracellular matrix composition immediatelyformed a filament with the diameter of the needle. If a blunt-tippedneedle is used, straight filaments can be formed while coiled filamentscan be formed with a bevel-tipped needle. Such filaments can be used asresorbable sutures.

Example 11 Lyophilization and Reconstitution of Solubilized ECMCompositions

Frozen small intestinal submucosa powder that had been prepared bycryogenic milling was centrifuged at 3000×g for 15 minutes and theexcess fluid was decanted. The powder (5% weight/volume) was digestedand solubilized in 0.01 N HCl containing 0.1% weight/volume pepsin forapproximately 72 hours at 4° C. The solubilized extracellular matrixcomposition was then centrifuged at 16,000×g for 30 minutes at 4° C. toremove the insoluble material. Aliquots of the solubilized extracellularmatrix composition were created and hydrochloric acid (12.1 N) was addedto create a range of concentrations from 0.01 to 0.5 N HCl.

Portions of the solubilized extracellular matrix composition weredialyzed (MWCO 3500) extensively against water and 0.01 M acetic acid todetermine the effects of these media on the lyophilization product.Aliquots of the solubilized extracellular matrix composition in 0.01 Macetic acid were created and glacial acetic acid (17.4 M) was added tocreate a range of concentrations from 0.01 to 0.5 M acetic acid. Thesolubilized extracellular matrix compositions were frozen using a dryice/ethanol bath and lyophilized to dryness. The lyophilizedpreparations were observed, weighed, and dissolved at 5 mg/ml in either0.01 N HCl or water. The dissolution and polymerization properties werethen evaluated. The results are shown in Tables 4-8.

TABLE 4 Gross appearance of solubilized extracellular matrixcompositions following lyophilization at various hydrochloric acidconcentrations. [HCl] (N) Appearance 0.01 Light, fluffy, homogenous,foam-like sheet; white to off-white in color; pliable 0.05 Slightlywrinkled and contracted, some inhomogeneities in appearance noted,slight brown tint, pliable to slightly friable in consistency 0.10Wrinkled, collapsed in appearance; inhomogeneities noted, some regional“melting” noted; significant brown tint; friable 0.25 Wrinkled,collapsed in appearance; increased inhomogeneities noted, increasedareas of regional “melting” noted; significant brown tint; friable 0.50Significant collapse and shrinkage of specimen, dark brown colorationthroughout; dark brown in color; friable

TABLE 5 Dissolution properties of solubilized extracellular matrixcompositions following lyophilization at various hydrochloric acidconcentrations. [HCl] (N) Reconstitution Reconstitution PropertiesMedium H₂O 0.01N HCl 0.01 Completely dissolved in Completely dissolvedin 20-30 minutes, pH 4 20-30 minutes, pH 2 0.05 Majority dissolved in 2Majority dissolved in hours; slight particulate 40 minutes; very slightnoted, pH 3-4 particulate noted, pH 2 0.1 Incomplete dissolutionIncomplete dissolution 0.25 Incomplete dissolution Incompletedissolution 0.5 Incomplete dissolution Incomplete dissolution

TABLE 6 Polymerization properties of solubilized extracellular matrixcompositions following lyophilization at various hydrochloric acidconcentrations. [HCl] (N) Reconstitution Polymerization PropertiesMedium H₂O 0.01N HCl 0.01 Polymerized within 20-30 Polymerized within10-20 minutes minutes 0.05 Weak gel noted at 45 Polymerized within 20-30minutes; significant lag minutes time in gelling 0.1 *No Polymerization*No Polymerization 0.25 *No Polymerization *No Polymerization 0.5 *NoPolymerization *No Polymerization

TABLE 7 Dissolution properties of solubilized extracellular matrixcompositions following lyophilization at various acetic acidconcentrations. [Acetic Reconstitution Properties Acid] (M)Reconstitution in H₂O Reconstitution in 0.01N HCl 0.01 Completelydissolved in 90 Completely dissolved in 90 minutes, pH 5 minutes, pH 1-20.05 Near complete dissolution Completely dissolved in 90 after 90minutes; small minutes, pH 1-2 particulate remained, pH 5 0.1 Completelydissolved in 90 Near complete dissolution in minutes, pH 5 90 minutes;small particulate, pH 1-2 0.25 Completely dissolved in 90 Completelydissolved in 90 minutes, pH 5 minutes, pH 1-2 0.5 Near completedissolution Completely dissolved in 90 after 90 minutes; small minutes,pH 1-2 particulate remained, pH 5

TABLE 8 Polymerization properties of solubilized extracellular matrixcompositions following lyophilization at various acetic acidconcentrations. [Acetic Acid] (M) Reconstitution PolymerizationProperties Medium H₂O 0.01N HCl 0.01 Polymerized within 5 10 minutes0.05 Polymerized within 5 10 minutes 0.1 Polymerized within 5 10 minutes0.25 Polymerized within 5 10 minutes 0.5 Polymerized within 5 10 minutes

These results show that lyophilization in HCl and reconstitution ofsolubilized extracellular matrix compositions in 0.01 N HCl to 0.05 NHCl or in water maintains the capacity of the components of thecompositions to polymerize. The results also show that lyophilization inacetic acid maintains the capacity of the components of the compositionsto polymerize when the composition is polymerized in water or HCl. Thesolubility rate is lyophilization from 0.01 N HCl>lyophilization from0.01 M acetic acid≧lyophilization from water.

Example 12 Preparation of Solubilized Submucosa Composition

1. Dissolution: of small intestinal submucosa (SIS) powder in aceticacid with pepsin

-   -   1.1. Preparation of acetic acid with pepsin        -   1.1.1. Prepare the desired volume of 0.5 M acetic acid            (typically 1 L; this requires 28.7 mL of 17.4 M glacial            acetic acid).        -   1.1.2. Add the desired mass of pepsin to achieve a 0.1% w/v            solution (typically 1 g, if 1 L of acetic acid is used).        -   1.1.3. Place the jar containing acetic acid and pepsin on a            stir plate and begin mixing.    -   1.2. Preparation of centrifuged SIS powder        -   1.2.1. Place SIS powder in 50 mL centrifuge tubes.        -   1.2.2. Centrifuge SIS powder at 3000×g for 15 minutes.        -   1.2.3. Open centrifuge tubes, pour off and dispose of            supernate.        -   1.2.4. Remove pellets from tubes. Measure out the desired            mass to achieve a 5% w/v solution (typically 50 g, if 1 L of            acetic acid was used). Previously prepared and frozen            material may be used, and excess centrifuged material may be            frozen for later use.    -   1.3. Add centrifuged SIS pellet material to acetic acid/pepsin        solution.    -   1.4. Cover and allow it to stir for 72 hours at 4° C.        2. Centrifugation of dissolved SIS    -   2.1. When removed from stirring, the SIS/pepsin solution should        appear viscous and somewhat uniform. Pour SIS/pepsin solution        into centrifuge jars. Balance jars as necessary.    -   2.2. This mixture should be centrifuged at 16,000×g for 30        minutes at 4° C. Refer to the operators manual or SOP for        instructions on using the centrifuge. If using the Beckman model        J2-21, use the JIO head at a speed of 9500 rpm.    -   2.3. Remove jars of SIS from centrifuge. Pour the supernatant        into a clean jar. Be careful not to disturb the pellet, and stop        pouring if the SIS begins to appear more white and creamy (this        is pellet material).        3. Dialysis of SIS in water and hydrochloric acid    -   3.1. Prepare dialysis tubing as follows:        -   3.1.1. Use dialysis tubing with MWCO 3500, diameter 29 lmll.            Handle dialysis tubing with gloves, and take care not to            allow it to contact foreign surfaces, as it may easily be            damaged.        -   3.1.2. Cut dialysis tubing to the necessary length.            (typically, 3 sections of about 45 cm).        -   3.1.3. Wet tubing in millipore water, and leave tubing in            the water until each piece is needed.        -   3.1.4. Do the following with each length of tubing:            -   3.1.4.1. Place a clip near one end of the tubing.            -   3.1.4.2. Holding the tubing to avoid contact with                foreign surfaces, use a pipette to fill the tubing with                SIS solution. Each piece of tubing should receive                roughly the same volume of SIS (for example, if three                lengths of tubing are used, measure one third of the                total volume into each).            -   3.1.4.3. Place a clip on the open end of the dialysis                tubing. Avoid leaving slack. The tube should be full and                taut.            -   3.1.4.4. Place the filled dialysis tubing in a container                of 0.01 M HCL with a stir bar.            -   3.1.4.5. Repeat the above steps to fill all lengths of                tubing.        -   3.1.5. Leave containers to stir at 4o C.    -   3.2. Details regarding changing the dialysis in 0.01 M HCl are        given below.        -   3.2.1. The 0.01 M HCl in the dialysis containers must be            changed several times. This should be done as follows:        -   3.2.2. After changing the 0.01 M HCl, another change should            not be done for at least two hours.        -   3.2.3. Change the 0.01 M HCl at least 10 times, over a            period of at least four days. This assumes a ratio of 200 mL            SIS to 6 L of 0.01 M HCl. If a higher ratio is used, more            changes may be necessary.        -   3.2.4. When changing 0.01 M HCl, do not leave dialysis bags            exposed in the air or on the counter. Use tongs or forceps            to move a dialysis bag directly from one container to            another. (It is okay to have multiple dialysis bags in one            container.) Dump the first container in the sink, then            refill it with millipore water. The dialysis bags can now be            placed in the newly filled container while the other            container or containers are changed.            4. Sterilization of SIS    -   4.1. Place dialysis bags of SIS in a solution of 0.18% Peracetic        acid/4.8% Ethanol. Leave to stir for two hours (more time may be        necessary).    -   4.2. Translocate dialysis bags to 0.01 M HCl, and continue        dialysis as before.        -   Continue for at least 2 days, changing HCl at least 3 times            daily.    -   4.3. When dialysis is complete, dialysis tubing filled with SIS        should be removed from the HCl.    -   4.4. Remove the clips. Cut open one end of the dialysis tubing        and pour SIS into a clean jar.    -   4.5. SIS should be refrigerated until use.        5. Lyophilization of SIS    -   5.1. Operating the Vertis Freezemobile        -   5.1.1. Make sure the condenser is free of any water. (The            condenser is the metal cylinder which opens on the front of            the lyophilizer.) Ensure that the black rubber collection            tubing attached to the bottom of the condenser is plugged.            This can be accessed by opening the grate on the front of            the lyophilizer.        -   5.1.2. Close the door of the condenser, the top of the            manifold, and all sample ports. If the door of the condenser            or the top of the manifold are not forming a good seal,            apply a small amount of vacuum grease to the rubber contact            surfaces.        -   5.1.3. Turn on the “Refrigerate” switch. The indicator on            the front of the lyophilizer will show a light beside            “Condenser” and beneath “On.” The light beneath “OK” will            not illuminate until the condenser is cooled. The condenser            temperature is indicated when the digital readout displays            “C1.”        -   5.1.4. When the “condenser” indicator light under “OK” is            illuminated, on the “Vacuum” switch. The indicator will show            a light beside “Condenser” and beneath “On.” The light            beneath “OK” will not turn on until the chamber is            sufficiently evacuated. The chamber pressure is indicated            when the digital readout displays “V 1.”        -   5.1.5. The rollers can be used for freezing a coat of            material on the inside surface of a jar. To use the rollers,            first ensure that the drain tube is plugged. (This can be            accessed through the door on the right side of the front of            the lyophilizer.) Using 100% Ethanol, fill the roller tank            to a level several millimeters above the top of the rollers.            Under-filling will cause ineffective cooling while            over-filling will allow ethanol to leak into the jars. The            temperature of ethanol bath is indicated when the digital            readout displays “T1.” This bath is cooled when the            “Refrigerate” switch is turned on. The “Rollers” switch            controls the turning of the rollers, and may be switched off            when no jar is on the rollers.    -   5.2. Lyophilizing SIS        -   5.2.1. Lyophilization jars, glass lids, and rubber gaskets            should be cleaned with ethanol. Allow ethanol to evaporate            completely before use. Mid-size jars, lids, and gaskets            (3-inch diameter) should be used to fit into the roller if            using the Virtis Freezemobile Jar lyophilization.        -   5.2.2. Pipette 75 mL of SIS solution into the lyophilization            jar. Place gasket and lid on jar.        -   5.2.3. Seal the jar by covering the openings with parafilm.            Note the small hole on the neck of the lid, which must be            covered.        -   5.2.4. Place the jar of SIS on the lyophilizer rollers for a            minimum of 2 hours.        -   5.2.5. Alternatively, the jar may be placed in a freezer            until all material is solid. In a −80° C. freezer, this            takes about 30 minutes.        -   5.2.6. Prepare a spigot on the lyophilizer by inserting a            glass cock with the tapered end out. The tapered end of the            cock should be coated with vacuum grease.        -   5.2.7. Remove the jar of SIS from the rollers (or freezer).            Place springs on the hooks to hold the jar and lid together.            Remove the parafilm and place the neck of the lid of the jar            over the cock. Rotate the jar so that the holes in the lid            and the cock do not align. The spigot can be rotated so that            the jar rests on the top surface of the lyophilizer.        -   5.2.8. Turn the valve switch so that it points toward the            jar of S1S.        -   5.2.9. More jars may be added to freeze-dry simultaneously,            but add jars one or two at a time. Wait until the vacuum            pressure falls to a reasonable range (e.g. 200 millitorr) to            ensure that the last jar is sealed before adding subsequent            jars.        -   5.2.10. Leave the jars under vacuum for at least 24 hours.        -   5.2.11. After lyophilization is complete, turn the switch On            the spigot to point away from the jar. This will allow air            into the jar.        -   5.2.12. Remove the jar from the cock.        -   5.2.13. Lyophilized material is not immediately used, it            should be stored in a dry environment. Use a large, sealable            container with Dri-Rite or another desiccant, and place            containers of lyophilized material therein.            6. Rehydration of lyophilized SIS    -   6.1. Place lyophilized SIS into a tube or jar.    -   6.2. Add the desired quantity of liquid (typically 0.01 N HCl)        to the container of SIS.    -   6.3. Mixing may be accelerated by shaking, stirring, etc. Store        container under refrigeration until dissolution of SIS is        complete.

Sterilization of Solubilized SIS by Dialysis Against Peracetic AcidContaining Solution

1. Dialyze solubilized SIS against a large reservoir containing 0.18%peracetic acid/4.8% ethanol in water. Dialysis time may vary dependingupon peracetic acid concentration, dialysis membrane molecular weightcut off, temperature, etc.

2. Transfer dialysis bags aseptically to reservoirs containing 0.01 NHCl. Dialyze extensively to reduce concentration of residual peraceticacid.

3. When dialysis is complete, dialysis tubing filled with solubilizedSIS should be removed from the dialysis tank aseptically.

4. Remove dialysis clips and pour or pipette solubilized SIS into asterile jar.

5. The disinfected solubilized SIS should be stored at 4° C. until use.

Sterilization of SIS by Direct Addition of Peracetic Acid to SISSolution

1. Add 100% Ethanol and 32 wt % peracetic acid to solubilized SIS tocreate a solution with final concentration of 0.18% peracetic acid/4.8%ethanol. Stir well and leave for two hours.

2. Place solubilized SIS in aseptic dialysis bags. Dialyze againststerile solution of 0.01 N HCl.

3. When dialysis is complete, dialysis tubing filled with solubilizedSIS should be removed from the dialysis tank aseptically.

4. Remove dialysis clips and pour or pipette solubilized SIS into asterile jar.

5. The disinfected solubilized SIS should be stored at 4° C. until use.

While the invention has been illustrated and described in full detail inthe drawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiment has been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. In addition, all publications cited in thisapplication are indicative of the abilities possessed by those ofordinary skill in the pertinent art and are hereby incorporated byreference in their entirety as if each had been individuallyincorporated by reference and fully set forth. U.S. patent applicationSer. No. 11/435,393 filed May 16, 2006, entitled “EngineeredExtracellular Matrices”, naming as inventor Sherry L. Yoytik-Harbin, isalso hereby incorporated herein by reference in its entirety.

What is claimed is:
 1. An extracellular matrix composition, comprising:a dry collagenous powder comprising an extracellular matrix hydrolysatematerial, wherein said extracellular matrix hydrolysate material iseffective to gel upon rehydration with an aqueous medium, and whereinsaid extracellular matrix hydrolysate material has been formed bydigesting an extracellular matrix material with a hydrochloric acidsolution containing an enzyme that digests the extracellular matrixmaterial.
 2. The composition of claim 1, wherein the extracellularmatrix hydrolysate material comprises components from vertebratesubmucosa tissue.
 3. The composition of claim 1, wherein theextracellular matrix hydrolysate material has been prepared by a processcomprising solubilizing an extracellular matrix composition withhydrochloric acid to produce a solubilized extracellular matrixmaterial, and lyophilizing the solubilized extracellular matrix materialto produce a lyophilized, solubilized extracellular matrix material thatis capable of polymerizing upon rehydration.
 4. A method of preparing alyophilized extracellular matrix composition, comprising the steps of:solubilizing an extracellular matrix composition with a hydrochloricacid solution containing an enzyme that digests the extracellular matrixcomposition to produce a solubilized extracellular matrix composition;and lyophilizing the solubilized extracellular matrix composition from asolution having a pH of about 6 or below to produce a lyophilized,solubilized extracellular matrix composition.
 5. The method of claim 4,wherein the extracellular matrix composition that is solubilizedcomprises vertebrate submucosa tissue or basement membrane tissue.
 6. Anextracellular matrix composition prepared by a process comprising:solubilizing an extracellular matrix composition with a hydrochloricacid solution containing an enzyme that digests the extracellular matrixcomposition, to form a solubilized composition of extracellular matrixcomponents; and lyophilizing the solubilized composition from ahydrochloric acid solution to produce a lyophilized, solubilizedextracellular matrix composition wherein the lyophilized, solubilizedcomposition, when dissolved at a concentration of 5 mg/ml in a 0.01 Mhydrochloric acid solution, is capable of polymerization within 30minutes.
 7. The composition of claim 6, wherein the enzyme is pepsin. 8.The composition of claim 6, wherein the hydrochloric acid solutioncontaining an enzyme that digests the extracellular matrix compositionis a 0.01 M to 0.05 M hydrochloric acid solution containing an enzymethat digests the extracellular matrix composition.
 9. The method ofclaim 4, wherein said lyophilizing is conducted in the absence of aceticacid.
 10. The method of claim 4, wherein said enzyme is pepsin.
 11. Themethod of claim 8, wherein the hydrochloric acid is at a concentrationof about 0.01 M.
 12. A composition comprising a lyophilizedextracellular matrix hydrolysate and an acid excipient comprisinghydrochloric acid, wherein the lyophilized extracellular hydrolysate,when dissolved at a concentration of 5 mg/ml in a 0.01 M hydrochloricacid solution, is capable of polymerization within 30 minutes.
 13. Thecomposition of claim 12 wherein the lyophilized extracellular matrixhydrolysate is prepared by lyophilizing the extracellular matrixhydrolysate from a 0.01 M to 0.05 M hydrochloric acid solution.
 14. Thecomposition of claim 13, wherein the lyophilized extracellular matrixhydrolysate is prepared by lyophilizing the extracellular matrixhydrolysate from an about 0.01 M hydrochloric acid solution.